GSM7474611: snATAC-seq of WT; Mus musculus; ATAC-seq
The frozen ventral horn of L3-L5 spinal cord was used for isolation the nuclei. The spinal cord was taken from the 9-month-old male mice, and quickly placed straightly in the embedding mold. Then the spinal cord was quickly frozen with OCT in liquid nitrogen. Removed the ventral white matter in microtome until the gray matter appears and removed the white matter on both sides carefully. Then taking the gray matter at the depth of 1/2 before the confluence of the gray matter of two sides and put the gray matter into the EP tube for liquid nitrogen quick freezing again. Then the nuclei were extracted For snRNA-seq, the single cell 3′ GEM, Library & Gel Bead Kit V3.1 (10× Genomics, 1000075) and Chromium Single Cell B Chip Kit (10× Genomics, 1000074) were used. To generate single-nuclei gel beads in emulsion, the nuclei suspension was loaded onto the Chromium single cell controller (10× Genomics). Then suspended the single nuclei in PBS (containing 0.04% BSA). Captured cells were lysed to release their RNA and barcoded through reverse transcription in individual GEMs. The reverse transcription was performed on a S1000TM Touch Thermal Cycler (Bio Rad) at 53°C for 45 min, followed by 85°C for 5 min. The cDNA was kept at 4°C and then amplified for sequencing. For snATAC-seq, incubating the nuclei with Tn5 transposase. Then the nuclei suspension was loaded into the Chromium microfluidic chip E with 10x Genomics reagents and barcoded with a 10x Genomics Chromium Controller (10x Genomics, Pleasanton, CA). DNA fragments were subsequently amplified, and the sequencing libraries were constructed with reagents from a Chromium Single Cell ATAC reagent kit (10x Genomics; PN-1000110, PN-1000156, PN-1000084) according to the manufacturer's instructions. After preliminary quantification and quality inspection, libraries were then pooled and loaded on an Illumina NovaSeq with 2 x 50 paired-end kits.